Thesis (Ph. D.)--University of Wisconsin--Madison, 1958. / Typescript. Abstracted in Dissertation abstracts, v. 19 (1958) no. 6, p. 1223. Section IA: Kinetics of the reversible Michaelis-Menten mechanism and the applicability of the steady state approximation, by W.G. Miller and R.A. Alberty. Includes Integrated rate equations for isotopic exchange in simple reversible reactions, by R.A. Alberty and W.G. Miller (reprinted from the Journal of chemical physics, v. 26 (1957) no. 5, p. 1231-1237) Studies of the enzyme fumarase: VI. Study of the incorporation of deuterium into L-malate during the reaction in deuterium oxide, by R.A. Alberty, W.G. Miller and H.F. Fisher, and The mechanism of the stereospecific enzymatic hydration of fumarate to L-malate, by T.C. Farrar ... [et al.] (reprinted from the Journal of the American Chemical Society, v. 79 (1957) p. 3973-3977, 3978-3980). Vita. eContent provider-neutral record in process. Description based on print version record. Includes bibliographical references.

One molecular form of fumarase from Ascaris suum was demonstrated by cellulose acetate electroporesis and isoelectric focusing. The enzyme was partially purified by ammonium sulfate fractionation and ion-exchange chromatography to a specific activity of 49 units per mg protein. Enzymatic assay of the partially purified by ammonium sulfate fractionation amd ion-exchange chromatography to a specific activity of 49 units per mg protein. Enzymatic assay of the partially purified preparation showed glyceraldehyde-3-phosphate dehydrongenase to be the major preparative contaminant.

Anaerobic fermentation has been an established technology ever since man started treating sewage. Recently this process has received increased attention because of its inherent ability to produce methane gas, which apart from solar energy, is the cleanest, most non-polluting source of energy. Methanobacterium thermoautotrophicum, a thermophilic bacterium, grows on CO(,2) as a source of carbon as well as electron acceptor, using hydrogen as an electron donor. Labeling studies carried out with ('14)C have shown a presence of partial reductive TCA cycle. In this work, the enzyme fumarate reductase, which belongs to this cycle, has been purified to homogeneity using various separation techniques. In keeping with the thermophilic character of the organism, fumarate reductase is extremely heat resistant. Incubation at 75(DEGREES)C for 24 hours led to an increase in purification. In contrast, the enzyme was found to be very sensitive to oxygen. The crude extract, when exposed to air, lost half of its activity within 20 minutes. Reducing agents were helpful in protecting against loss of enzymatic activity provided that a strict anaerobic atmosphere was maintained. For this reason, the entire purification was performed inside a Freter-type anaerobic chamber using reducing agents. The molecular weight of the native fumarate reductase, as determined by Sephacryl S-300 gel exclusion chromatography, was found to be approximately 80,000. SDS polyacrylamide gel electrophoresis data suggested that the enzyme is a tetramer. Treatment with sulfhydyl reagents as well as Cu('++) caused loss in fumarate reductase activity, indicating that the enzyme contains at least one sulfhydryl group which is important to its activity. The UV/Visible spectrum of fumarate reductase did not reveal the presence of a flavin moiety as a cofactor. Both UV/Visible and fluorescence spectra of fumarate reductase from M. thermoautotrophicum instead, indicated the presence of an unusual cofactor, which could be similar to either tetrahydromethanopterin or F(,420).

Trypanosoma cruzi é um protozoário flagelado que ao infectar seres humanos causa a doença de Chagas, uma doença tropical negligenciada que afeta milhões de pessoas no mundo todo. As fumarato hidratases (FH), ou fumarases, são enzimas que catalisam a reação estéreo-específica reversível de hidratação do fumarato em S-malato, e foram recentemente consideradas essenciais para a viabilidade do parasito Trypanosoma brucei, sugerindo seu potencial como alvo macromolecular para o desenvolvimento de novos fármacos tripanocidas. O presente trabalho visou à caracterização funcional, bioquímica, biofísica e estrutural das fumarases de T. cruzi (TcFHs) e humana (HsFH) de forma a avaliar o papel das TcFHs para o parasito Trypanosoma cruzi, mapear o mecanismo de ação e identificar as diferenças entre TcFHs e a enzima humana de forma a serem exploradas no planejamento de inibidores seletivos às fumarases do parasito. Análise das sequências mostrou que TcFHs pertencem à classe I das fumarases (enzimas diméricas dependentes de ferro) e não são homólogas à HsFH que pertence a classe II (tetraméricas independentes de ferro). Estudos de localização celular confirmaram a existência de duas fumarases em T. cruzi, uma citosólica (TcFHc) e uma mitocondrial (TcFHm), e experimentos de nocaute gênico sugeriram que essas enzimas são essências para o parasito. A caracterização cinética das enzimas TcFHc, TcFHm e HsFH mostrou que as fumarases de T. cruzi são sensíveis ao oxigênio enquanto a enzima humana se mantém ativa em condições aeróbicas. Estudos de ressonância eletrônica paramagnética mostraram a presença de um cluster de ferro-enxofre, sensível a oxidação por oxigênio, envolvido no mecanismo enzimático das enzimas TcFHs. Modelos estruturais das TcFHs, construídos por homologia à estrutura cristalográfica da fumarase de Leishmania major, foram comparados à estrutura cristalográfica obtida para a fumarase humana e as diferenças entre as duas estruturas foram utilizadas no planejamento de ligantes seletivos às fumarases do parasito. O ligante planejado inibiu a fumarase citosólica de T. cruzi na faixa de 1 ?M e não apresentou efeito na atividade da enzima humana. Testes in vivo demonstraram o efeito tripanocida do inibidor provavelmente por interferir na produção de ATP pela mitocôndria do T. cruzi. Os resultados obtidos com o desenvolvimento desse projeto apresentam uma proposta inovadora no desenvolvimento de novas terapias contra a doença de Chagas, o uso da enzima fumarase como alvo macromolecular, assim como apresenta um inibidor potente e seletivo para a enzima do parasito a ser utilizado como protótipo no desenvolvimento de fármacos contra Trypanosoma cruzi. A síntese de moléculas análogas ao inibidor de forma a melhorar suas propriedades farmacológicas encontra-se em andamento / Trypanosoma cruzi is a flagellate protozoan parasite that infects humans and causes Chagas disease, a tropical neglected disease that affects millions of people worldwide. Fumarate hydratases (FH), or fumarases, are enzymes responsible for the reversible stereo-specific hydration of fumarate into S-malate, and were recently considered to be essential to Trypanosoma brucei viability, suggesting, therefore, a potential role for FHs as macromolecular targets to the drug development against trypanosomatids. The present work focused on the functional, biochemical, biophysical and structural characterization of T. cruzi fumarases (TcFHs) and human fumarase (HsFH) to evaluate TcFHs role for T. cruzi, map the reaction mechanism and identify and exploit differences between the parasite and host enzymes in order to design selective inhibitors to the parasite enzyme. Sequence analysis revealed that TcFHs belong to class I fumarases (dimeric and iron-sulfur containing enzymes) and are not homologous to HsFH which belongs to class II fumarases (tetrameric iron independent enzymes). Cellular sub-localization studies confirmed the presence of a cytosolic and a mitochondrial fumarases in T. cruzi and gene knockout experiments suggested TcFHs are essential to the parasite. The kinetic characterization showed that TcFHs activity is highly sensitive to oxygen whereas HsFH activity remained stable in aerobic conditions. Electron paramagnetic experiments further revealed the presence of an iron-sulfur cluster highly sensitive to oxidation and involved in the catalytic mechanism in both TcFHm and TcFHc. TcFHs structural models, built by homology modeling using the Leishmania major fumarase crystal structure as template, were compared to the HsFH crystal structure and the differences were used to design a selective ligand to the parasite fumarases. The designed ligand showed to inhibit TcFHc with an IC50 of 1 ?M and showed no effect on the human fumarase activity. In vivo assays using T. cruzi epimastigotes demonstrated the trypanocidal effect of the designed inhibitor probably caused by stalling ATP production. The results obtained with the development of this project represent an innovative proposal on the development of new therapies against Chagas disease, the use of fumarase enzyme as a macromolecular target, as well as present a potent and selective inhibitor to the parasite enzyme to be further used as a prototype in the development of drugs against Chagas disease. The synthesis of inhibitor analogues with optimized pharmacological properties are currently in progress.

Trypanosoma cruzi é um protozoário flagelado que ao infectar seres humanos causa a doença de Chagas, uma doença tropical negligenciada que afeta milhões de pessoas no mundo todo. As fumarato hidratases (FH), ou fumarases, são enzimas que catalisam a reação estéreo-específica reversível de hidratação do fumarato em S-malato, e foram recentemente consideradas essenciais para a viabilidade do parasito Trypanosoma brucei, sugerindo seu potencial como alvo macromolecular para o desenvolvimento de novos fármacos tripanocidas. O presente trabalho visou à caracterização funcional, bioquímica, biofísica e estrutural das fumarases de T. cruzi (TcFHs) e humana (HsFH) de forma a avaliar o papel das TcFHs para o parasito Trypanosoma cruzi, mapear o mecanismo de ação e identificar as diferenças entre TcFHs e a enzima humana de forma a serem exploradas no planejamento de inibidores seletivos às fumarases do parasito. Análise das sequências mostrou que TcFHs pertencem à classe I das fumarases (enzimas diméricas dependentes de ferro) e não são homólogas à HsFH que pertence a classe II (tetraméricas independentes de ferro). Estudos de localização celular confirmaram a existência de duas fumarases em T. cruzi, uma citosólica (TcFHc) e uma mitocondrial (TcFHm), e experimentos de nocaute gênico sugeriram que essas enzimas são essências para o parasito. A caracterização cinética das enzimas TcFHc, TcFHm e HsFH mostrou que as fumarases de T. cruzi são sensíveis ao oxigênio enquanto a enzima humana se mantém ativa em condições aeróbicas. Estudos de ressonância eletrônica paramagnética mostraram a presença de um cluster de ferro-enxofre, sensível a oxidação por oxigênio, envolvido no mecanismo enzimático das enzimas TcFHs. Modelos estruturais das TcFHs, construídos por homologia à estrutura cristalográfica da fumarase de Leishmania major, foram comparados à estrutura cristalográfica obtida para a fumarase humana e as diferenças entre as duas estruturas foram utilizadas no planejamento de ligantes seletivos às fumarases do parasito. O ligante planejado inibiu a fumarase citosólica de T. cruzi na faixa de 1 ?M e não apresentou efeito na atividade da enzima humana. Testes in vivo demonstraram o efeito tripanocida do inibidor provavelmente por interferir na produção de ATP pela mitocôndria do T. cruzi. Os resultados obtidos com o desenvolvimento desse projeto apresentam uma proposta inovadora no desenvolvimento de novas terapias contra a doença de Chagas, o uso da enzima fumarase como alvo macromolecular, assim como apresenta um inibidor potente e seletivo para a enzima do parasito a ser utilizado como protótipo no desenvolvimento de fármacos contra Trypanosoma cruzi. A síntese de moléculas análogas ao inibidor de forma a melhorar suas propriedades farmacológicas encontra-se em andamento / Trypanosoma cruzi is a flagellate protozoan parasite that infects humans and causes Chagas disease, a tropical neglected disease that affects millions of people worldwide. Fumarate hydratases (FH), or fumarases, are enzymes responsible for the reversible stereo-specific hydration of fumarate into S-malate, and were recently considered to be essential to Trypanosoma brucei viability, suggesting, therefore, a potential role for FHs as macromolecular targets to the drug development against trypanosomatids. The present work focused on the functional, biochemical, biophysical and structural characterization of T. cruzi fumarases (TcFHs) and human fumarase (HsFH) to evaluate TcFHs role for T. cruzi, map the reaction mechanism and identify and exploit differences between the parasite and host enzymes in order to design selective inhibitors to the parasite enzyme. Sequence analysis revealed that TcFHs belong to class I fumarases (dimeric and iron-sulfur containing enzymes) and are not homologous to HsFH which belongs to class II fumarases (tetrameric iron independent enzymes). Cellular sub-localization studies confirmed the presence of a cytosolic and a mitochondrial fumarases in T. cruzi and gene knockout experiments suggested TcFHs are essential to the parasite. The kinetic characterization showed that TcFHs activity is highly sensitive to oxygen whereas HsFH activity remained stable in aerobic conditions. Electron paramagnetic experiments further revealed the presence of an iron-sulfur cluster highly sensitive to oxidation and involved in the catalytic mechanism in both TcFHm and TcFHc. TcFHs structural models, built by homology modeling using the Leishmania major fumarase crystal structure as template, were compared to the HsFH crystal structure and the differences were used to design a selective ligand to the parasite fumarases. The designed ligand showed to inhibit TcFHc with an IC50 of 1 ?M and showed no effect on the human fumarase activity. In vivo assays using T. cruzi epimastigotes demonstrated the trypanocidal effect of the designed inhibitor probably caused by stalling ATP production. The results obtained with the development of this project represent an innovative proposal on the development of new therapies against Chagas disease, the use of fumarase enzyme as a macromolecular target, as well as present a potent and selective inhibitor to the parasite enzyme to be further used as a prototype in the development of drugs against Chagas disease. The synthesis of inhibitor analogues with optimized pharmacological properties are currently in progress.

The aim of this research was to identify the mechanisms that underpin changes in respiratory capacity during acclimation to temperature. Dark respiration, enzyme activities and leaf ultrastructure were measured from ryegrass (Lolium perenne) in controlled environmental chambers and two species of native grass (Chionochloa rubra & C. pallens) growing at different altitudinal ranges on Mount Hutt, Canterbury, New Zealand. The overall hypothesis was that the changes in both mitochondrial numbers and enzyme activity underpin the greater respiratory capacity observed in response to decreasing temperatures. Gas exchange measurements were carried out to measure rates of dark respiration (Rd) in leaves of both ryegrass and tussocks. Respiratory homeostasis (full acclimation) was achieved in ryegrass leaves but only partial acclimation in both species of tussock plants. Dark respiration rates for warm-grown ryegrass were greatly reduced compared to cool-grown grasses. Rd was lower for C. rubra growing at the base of the mountain (450m) compared to plants at a higher altitude (1060m). The dark respiration rates were also lower for C. pallens growing at 1070m than at 1600m. When comparing Rd between high and low altitude plants, it was significantly lower in low altitude plants at 450m than at 1600m. Oxygen consumption was measured in intact leaves and roots, crude mitochondria and isolated mitochondria from ryegrass to investigate whether a change in respiratory capacity was involved with changes in Rd. Mitochondrial respiratory capacity was slightly reduced in warm leaves and roots (not significantly). The respiratory capacity results from isolated mitochondria for C. rubra (at 450m and 1060m) and C. pallens (at 1070m and 1600m) were consistent with the hypothesis that plants from warm sites have lower respiratory capacity in comparison to plants from cool sites. Based on these results and those of previous studies, it was concluded that respiratory flux for any given temperature is not simply determined by maximal capacities of the respiratory apparatus but rather a combination of the availability of substrate supply, the demand for respiratory products (i.e. ATP) and/or the maximal capacity of respiratory enzymes. Utilizing transmission electron micrographs, it was found that mitochondria were significantly less abundant in warm-grown than cool-grown ryegrass mesophyll cells. Mitochondria dimensions increased slightly between the cool and warm treatment. At lower altitudes (C. rubra), there was a significant decrease in mitochondria numbers with decreasing elevation. At higher altitudes (C. pallens), there was no noticeable change in mitochondria numbers between 1070m and 1600m. It was concluded that mitochondrial abundance for the controlled and field experiments, and mitochondrial sizes in the field, were associated with changes in Rd. The maximal activities of fumarase and succinate dehydrogenase (SDH) in isolated mitochondria from leaves of ryegrass and tussock were measured spectrophotometrically. The results in the controlled experiment indicate that enzymes other than fumarase and SDH could be responsible for the increased respiratory capacity observed in cold acclimated leaves of ryegrass. However, fumarase maximal activity was significantly reduced in C. rubra at low altitude compared with C. pallens growing at high altitude - this suggests that it may be involved in the differences in respiratory capacity and Rd between the two sites. Succinate dehydrogenase did not differ significantly in response to altitude. The large difference between the two field sites for fumarase activity is comparable to the large difference in Rd and reduction in mitochondrial abundance and dimensions seen between the two sites. This supports the overall hypothesis that cool-grown plants keep up with energy demands at low temperatures by increasing enzyme concentrations/capacity. The results of this study are supportive of the hypothesis that growth in low altitudes and warm conditions will result in the reduction of Rd as a consequence of: (1) temperature sensitivity of the respiratory apparatus, resulting in the reduction of the respiratory capacities of mitochondria; (2) a reduction in mitochondria size and numbers; and as a consequence of this (3) a reduction in the activities of mitochondrial enzymes. However, these responses are species specific and vary according to the range of temperatures experienced by plants in the field and controlled environments.

<p>In eukaryotic cells, DNA is packaged into a structure named chromatin which contains DNA and proteins. Nucleosomes are building blocks of chromatin and contain DNA wrapped around a histone octamer. Chromatin modifications (histone post-translational modifications and histone variants) play central roles in various cellular processes including gene expression and DNA damage response. Chromatin modifying enzymes use metabolites as co-substrates and co-factors, and changes in metabolic pathways and metabolite availability affects chromatin modifications and chromatin-associated functions. Moreover, recent studies have uncovered direct roles of metabolic enzymes in chromatin-associated functions. Fumarase, a TCA cycle enzyme that catalyzes the reversible conversion of fumarate to malate in mitochondria (a hydration reaction), is an example of an enzyme with dual functions in metabolism and genome integrity. Cytoplasmic fraction of yeast fumarase, Fum1p, localizes to the nucleus and promotes growth upon DNA damage. Fum1p promotes homologous recombination by enhancing DNA end resection. Human fumarase is involved in DNA repair by non-homologous end joining. Here, we provide evidence that yeast Fum1p and the histone variant Htz1p are also involved in DNA replication stress response and DNA repair by non-homologous end joining (NHEJ). Using mutants lacking the histone variant <i>HTZ1</i>, we show that high cellular levels of fumarate, by deletion of <i>FUM1</i> or addition of exogenous fumarate, suppressed the sensitivity to DNA replication stress by modulation of activity of Jhd2p. This suppression required sensors and mediators of the intra-S phase checkpoint, but not factors involved in the processing of replication intermediates. These results imply that high cellular levels of fumarate can confer resistance to DNA replication stress by bypassing or complementing the defects caused by loss of <i>HTZ1</i> and replication fork processing factors. We also show that upon induction of DSBs, exogenous fumarate conferred resistance to mutants with defects in NHEJ, early steps of homologous recombination (DNA end resection pathway) or late steps of homologous recombination (strand invasion and exchange). Taken together, these results link the metabolic enzyme fumarase and the metabolite fumarate to DNA damage response and show that modulation of DNA damage response by regulating activity of chromatin modifying enzymes is a plausible pathway linking metabolism and nutrient availability to chromatin-associated functions like genome integrity.<br><a></a></p>

The aim of this research was to identify the mechanisms that underpin changes in respiratory capacity during acclimation to temperature. Dark respiration, enzyme activities and leaf ultrastructure were measured from ryegrass (Lolium perenne) in controlled environmental chambers and two species of native grass (Chionochloa rubra & C. pallens) growing at different altitudinal ranges on Mount Hutt, Canterbury, New Zealand. The overall hypothesis was that the changes in both mitochondrial numbers and enzyme activity underpin the greater respiratory capacity observed in response to decreasing temperatures. Gas exchange measurements were carried out to measure rates of dark respiration (Rd) in leaves of both ryegrass and tussocks. Respiratory homeostasis (full acclimation) was achieved in ryegrass leaves but only partial acclimation in both species of tussock plants. Dark respiration rates for warm-grown ryegrass were greatly reduced compared to cool-grown grasses. Rd was lower for C. rubra growing at the base of the mountain (450m) compared to plants at a higher altitude (1060m). The dark respiration rates were also lower for C. pallens growing at 1070m than at 1600m. When comparing Rd between high and low altitude plants, it was significantly lower in low altitude plants at 450m than at 1600m. Oxygen consumption was measured in intact leaves and roots, crude mitochondria and isolated mitochondria from ryegrass to investigate whether a change in respiratory capacity was involved with changes in Rd. Mitochondrial respiratory capacity was slightly reduced in warm leaves and roots (not significantly). The respiratory capacity results from isolated mitochondria for C. rubra (at 450m and 1060m) and C. pallens (at 1070m and 1600m) were consistent with the hypothesis that plants from warm sites have lower respiratory capacity in comparison to plants from cool sites. Based on these results and those of previous studies, it was concluded that respiratory flux for any given temperature is not simply determined by maximal capacities of the respiratory apparatus but rather a combination of the availability of substrate supply, the demand for respiratory products (i.e. ATP) and/or the maximal capacity of respiratory enzymes. Utilizing transmission electron micrographs, it was found that mitochondria were significantly less abundant in warm-grown than cool-grown ryegrass mesophyll cells. Mitochondria dimensions increased slightly between the cool and warm treatment. At lower altitudes (C. rubra), there was a significant decrease in mitochondria numbers with decreasing elevation. At higher altitudes (C. pallens), there was no noticeable change in mitochondria numbers between 1070m and 1600m. It was concluded that mitochondrial abundance for the controlled and field experiments, and mitochondrial sizes in the field, were associated with changes in Rd. The maximal activities of fumarase and succinate dehydrogenase (SDH) in isolated mitochondria from leaves of ryegrass and tussock were measured spectrophotometrically. The results in the controlled experiment indicate that enzymes other than fumarase and SDH could be responsible for the increased respiratory capacity observed in cold acclimated leaves of ryegrass. However, fumarase maximal activity was significantly reduced in C. rubra at low altitude compared with C. pallens growing at high altitude - this suggests that it may be involved in the differences in respiratory capacity and Rd between the two sites. Succinate dehydrogenase did not differ significantly in response to altitude. The large difference between the two field sites for fumarase activity is comparable to the large difference in Rd and reduction in mitochondrial abundance and dimensions seen between the two sites. This supports the overall hypothesis that cool-grown plants keep up with energy demands at low temperatures by increasing enzyme concentrations/capacity. The results of this study are supportive of the hypothesis that growth in low altitudes and warm conditions will result in the reduction of Rd as a consequence of: (1) temperature sensitivity of the respiratory apparatus, resulting in the reduction of the respiratory capacities of mitochondria; (2) a reduction in mitochondria size and numbers; and as a consequence of this (3) a reduction in the activities of mitochondrial enzymes. However, these responses are species specific and vary according to the range of temperatures experienced by plants in the field and controlled environments.

Introduction: Uterine fibroids are the most common benign tumours of female genital tract with the peak incidence in the 4th and 5th decennium. The aetiology of uterine fibroids still remains poorly understood. Genetic factors play undisputed role in the onset of uterine fibroids. Up to date numerous gene mutations were identified in certain percentage of patients with uterine fibroids. One of the candidate genes is Fumarate hydratase gene (FH). Heterozygous germiline mutations of FH cause two hereditary syndromes: Multiple smooth muscle tumours of the skin and uterus (MCUL1)/ Hereditary leiomyomatosis and renal cell cancer syndrome (HLRCC) characterised by leiomyomata of the skin, early onset uterine fibroids between 20-30 years of age and renal papillary carcinoma. The aim of our thesis was to identify the frequency of FH mutations in patients with early onset sporadic uterine fibroids. Methods: Patients with the diagnosis of uterine fibroids up to the age of 30 years were enrolled in the study. Control group consisted of patients with absence of uterine fibroids. Activities of Fumarate hydratase and control protein Citrate synthase were measured in lymphocytes and compared to the results obtained from the healthy controls. Mutation analysis of FH gene was performed. Activity of Fumarate...

京都大学 / 0048 / 新制・課程博士 / 博士(医学) / 甲第21954号 / 医博第4496号 / 新制||医||1037(附属図書館) / 京都大学大学院医学研究科医学専攻 / (主査)教授 渡邉 大, 教授 伊佐 正, 教授 椛島 健治 / 学位規則第4条第1項該当 / Doctor of Medical Science / Kyoto University / DFAM